Plasma angiotensin-(1-8) octapeptide measurement to assess acute angiotensin-converting enzyme inhibition with captopril administered parenterally to normal subjects.

The blood pressure (BP), heart rate (HR), and humoral effects of single intravenous (i.v.) doses of the angiotensin-converting enzyme (ACE) inhibitor captopril was investigated in five normotensive healthy volunteers. Each subject received at 1-week intervals a bolus dose of either captopril (1, 5, and 25 mg) or its vehicle. The study was conducted in a single-blind fashion, and the order of treatment phases was randomized. The different doses of captopril had no acute effect on BP and HR. They induced a dose-dependent decrease in plasma ACE activity and plasma angiotensin II levels. The angiotensin-(1-8) octapeptide was isolated by solid-phase extraction and high-performance liquid chromatography (HPLC) prior to radioimmunoassay (RIA). All three doses of captopril reduced circulating angiotensin II levels within 15 min of drug administration. Only with the 25-mg dose was the angiotensin II concentration below the detection limit at 15 min and still significantly reduced 90 min after drug administration. Simultaneous and progressive decreases in plasma aldosterone levels were observed both with ACE inhibition and during vehicle injection, but the relative fall was more pronounced after captopril administration. No adverse reaction was noticed. These results demonstrate that captopril given parenterally blocks the renin-angiotensin system in a dose-dependent manner. Only with the dose of 25 mg was the inhibition of plasma-converting enzyme activity and the reduction of plasma angiotensin II sustained for at least 1 1/2 h.

Angiotensin II is an endogenous neurotransmitter for rat and human mesenteric resistance blood vessels

Angiotensin II (Ang II) is one of the most potent vasoconstrictors. We document here the innervation of rat and human mesenteric resistance arteries (MRA) by angiotensinergic neurons of the rat and human sympathetic coeliac ganglia. Angiotensinogen (Ang-N)-mRNA and angiotensin converting enzyme-mRNA but no renin-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia. In the same extracts, cathepsin D-mRNA was detected: This protease also cleaves Ang I from Ang-N and could therefore account for the generation of neuronal Ang peptides in the absence of renin. In situ hybridization confirmed the presence of Ang-N-mRNA in the cytoplasm of rat coeliac ganglia. By using solid-phase extraction, high performance liquid chromatography and subsequent radioimmunoassay, Ang II and its metabolites were detected in rat and also in human coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia neurons and their projections innervating MRA. In addition, segmental angiotensinergic innervation of MRA was also observed. By means of confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings could indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally in MRA.

Circulating estrogens and progesterone during primiparous pregnancies and risk of maternal breast cancer.

Pregnancy reduces maternal risk of breast cancer in the long term, but the biological determinants of the protection are unknown. Animal experiments suggest that estrogens and progesterone could be involved, but direct human evidence is scant. A case-control study (536 cases and 1,049 controls) was nested within the Finnish Maternity Cohort. Eligible were primiparous women who delivered at term a singleton offspring before age 40. For each case, two individually matched controls by age (±6 months) and date of sampling (±3 months) were selected. Estradiol, estrone and progesterone in first-trimester serum were measured by high-performance liquid chromatography tandem mass spectrometry and sex-hormone binding globulin (SHBG) by immunoassay. Odds ratios (OR) and 95% confidence intervals (CI) were estimated through conditional logistic regression. In the whole study population there was no association of breast cancer with any of the studied hormones. In analyses stratified by age at diagnosis, however, estradiol concentrations were positively associated with risk of breast cancer before age 40 (upper quartile OR, 1.81; CI, 1.08-3.06), but inversely associated with risk in women who were diagnosed ≥age 40 (upper quartile OR, 0.64; CI, 0.40-1.04), p(interaction) 0.004. Risk estimates for estrone mirrored those for estradiol but were less pronounced. Progesterone was not associated with risk of subsequent breast cancer. Our results provide initial evidence that concentrations of estrogens during the early parts of a primiparous pregnancy are associated with maternal risk of breast cancer and suggest that the effect may differ for tumors diagnosed before and after age 40.

The bioavailability of bromazepam, omeprazole and paracetamol given by nasogastric feeding tube.

AIMS: To characterize and compare the pharmacokinetic profiles of bromazepam, omeprazole and paracetamol when administered by the oral and nasogastric routes to the same healthy cohort of volunteers. METHODS: In a prospective, monocentric, randomized crossover study, eight healthy volunteers received the three drugs by the oral (OR) and nasogastric routes (NT). Sequential plasma samples were analyzed by high-performance liquid chromatography-UV, pharmacokinetic parameters (Cmax, AUC(0-infinity), t(1/2), k(e), tmax) were compared statistically, and Cmax, AUC(0-infinity) and t(max) were analyzed for bioequivalence. RESULTS: A statistically significant difference was seen in the AUC(0-infinity) of bromazepam, with nasogastric administration decreasing availability by about 25%: AUC(OR) = 2501 ng mL(-1) h; AUC(NT) = 1855 ng mL(-1) h (p < 0.05); ratio (geometric mean) = 0.74 [90% confidence interval (CI) 0.64-0.87]. However, this does not appear to be clinically relevant given the usual dosage range and the drug's half-life (approx. 30 h). A large interindividual variability in omeprazole parameters prevented any statistical conclusion from being drawn in terms of both modes of administration despite their similar average profile: AUC(OR) = 579 ng mL(-1) h; AUC(NT) = 587 ng mL(-1) h (p > 0.05); ratio (geometric mean) = 1.01 (90% CI 0.64-1.61). An extended study with a larger number of subjects may possibly provide clearer answers. The narrow 90% confidence limits of paracetamol indicate bioequivalence: AUC(OR) = 37 microg mL(-1) h; AUC(NT) = 41 microg mL(-1) h(p > 0.05); ratio (geometric mean) = 1.12 (90% CI 0.98-1.28). CONCLUSION: The results of this study show that the nasogastric route of administration does not appear to cause marked, clinically unsuitable alterations in the bioavailability of the tested drugs.

Loco-regional administration of anticancer agents : isolated lung perfusion with doxorubicin hypoxic abdominal stop-flow perfusion with gemcitabine

SUMMARY Regional drug delivery is an approach designed to improve the selectivity of anticancer chemotherapy. The advantage of regional treatments lies in increasing the drug concentration in the affected organ, while the rest of the organism is spared, thus improving efficacy and limiting treatment toxicity. The goal of this thesis was to assess the distribution throughout the body and the disposition (pharmacokinetics) of two anticancer agents, doxorubicin and gemcitabine, administered by two different regional administration modalities: isolated lung perfusion (ILP) for pulmonary metastases from soft tissue sarcomas and abdominal stop-flow hypoxic perfusion for advanced pancreatic cancers, respectively. For this purpose, two high-performance liquid chromatography methods were developed and validated. The first enabled the determination of doxorubicin in four different biological matrices: serum, reconstituted effluent, tissues with low levels of doxorubicin and tissues with high levels of doxorubicin. The second allows the analysis of gemcitabine and its principal metabolite dFdU in plasma. The administration of doxorubicin by ILP was studied in three preclinical studies (one on pigs and two on rats). It was first shown that, regardless of the administration mode, doxorubicin was not homogeneously distributed throughout the lung and that some regions remained out of reach. Secondly, it was demonstrated that doxorubicin did not adequately reach the tumours despite very high levels found in the lung. Finally, an attempt to enhance the doxorubicin tumoural uptake by pharmacologic modulation using two P-glycoprotein inhibitors, cyclosporin and valspodar, was unsuccessful. The last part of this work involves the administration of gemcitabine by abdominal stop-flow as a part of a phase I clinical trial in patients with advanced pancreatic disease or resistant malignant ascites. The study has demonstrated that the regional exposure to gemcitabine was increased while the exposure of the entire organism was similar to standard intravenous administrations. From a toxicological perspective, the procedure was rather well tolerated. However, even if no clinical response is expected from a phase I study, no hints of clinical responses were unfortunately observed. In conclusion, even if loco-regional therapies may afford the pharmacological advantage of increasing anticancer drug levels at the tumour site, further studies of these investigational treatment modalities are warranted to ascertain whether they can provide a significant improvement of the cancer therapy for patients, in terms of treatment tolerability, improved responses and survival rates. RÉSUMÉ L'administration locorégionale d'agents anticancéreux est une approche destinée à augmenter la sélectivité du traitement. L'avantage des traitements régionaux repose sur le fait que la concentration du médicament cytostatique est augmentée dans l'organe où est localisée la tumeur, alors que le reste de l'organisme est épargné, améliorant ainsi en théorie l'efficacité du traitement et en limitant sa toxicité. Le but de ce travail de thèse avait pour objectif de préciser, la pharmacocinétique au sein de l'organisme de deux agents anticancéreux, la doxorubicine et la gemcitabine, administrés par deux types de perfusions loco-régionales: la perfusion isolée du poumon (ILP) pour les métastases pulmonaires de sarcomes des tissus mous, et la perfusion hypoxique (stop-flow) abdominale pour les cancers avancés du pancréas. Dans cette optique, deux méthodes de chromatographie liquide à haute performance ont été développées et validées. La première permet le dosage de la doxorubicine dans quatre milieux biologiques: le sérum, l'effluent reconstitué, ainsi que des tissus contenant des concentrations faibles et élevées en doxorubicine. La seconde méthode permet le dosage dans le plasma de la gemcitabine et de son principal métabolite, le dFdU. L'administration de doxorubicine par ILP a été étudiée dans trois études précliniques (une chez le porc et deux chez le rat). Il a été montré, dans un premier temps, que la doxorubicine n'était pas distribuée de façon homogène au sein du poumon, quel que soit son mode d'administration. Dans un deuxième temps, il a été démontré que le médicament n'atteignait pas les tumeurs de façon adéquate, malgré des concentrations très élevées au sein du tissu pulmonaire. Finalement, une tentative d'augmenter la pénétration tumorale de la doxorubicine par une modulation pharmacologique de la P-glycoprotéine en utilisant la cyclosporine et le valspodar n'a pas abouti. La dernière partie de ce travail concernait l'administration de gemcitabine par stop-flow abdominal dans le cadre d'une étude clinique de phase I menée auprès de patients atteints de cancers avancés du pancréas ou d'ascites malignes réfractaires. Cette étude a démontré que l'exposition régionale à la gemcitabine était augmentée, alors que l'exposition de l'organisme était similaire à une administration de dose standard par voie intraveineuse. D'un point de vue toxicologique la procédure fut relativement bien tolérée. Cependant, même s'il n'est pas attendu de réponses cliniques dans une étude de phase I, aucun signe de réponse au traitement n'a pu être malheureusement observé. En conclusion, même si les thérapies loco-régionales présentent -en théorie- l'avantage pharmacologique d'augmenter les taux du médicaments anticancéreux sur le site de la tumeur, d'autres études précliniques et cliniques sont nécessaires pour démontrer que ces nouvelles modalités de traitement, de nature investigationelle à présent, apportent une réelle amélioration pour la prise en charge des patients cancéreux, en terme de tolérance au traitement et de l'augmentation des taux de réponses et de survie.

Immunomodulation nutritionnelle chez les patients atteints de mucoviscidose

SUMMARY Pulmonary Pulmonary disease is the primary cause of morbidity and mortality in cystic fibrosis patients (CF). Airways of CF patients are early colonized by various bacteriae, and an intense inflammatory response participates to airways destruction. Accumulation of neutrophils releasing proteolytic enzymes and free radicals induce progressive lung tissue destruction in CF. Among several inflammatory mediators implicated in this process, chemotactic factors such as leukotriene B4 (LTB4), product of arachidonic omega-6 polyunsaturated fatty acid (PUFA), plays an important role. Many anti-inflammatory therapies including corticosteroids, ibuprofen, macrolides, antioxidants and antiproteinases have been proposed in CF over the last 20 years. In complement to these various approaches, dietary supplementation with polyunsaturated fatty acids (PUFA) omega-3, known to favor the synthesis of less inflammatory leukotriene B5 (LTB5), could also represent a potential. therapy. The objective of this thesis was to assess the impact of this nutritional approach on several CF neutrophil functions. In addition, we have also examined the influence of this approach on various clinical parameters, to assess the feasibility of future studies specifically oriented towards clinical effects. To that endeavour, a high performance liquid chromatography method has been developed and validated, allowing the simultaneous determination of LTB4 and LTB5 produced by stimulated human polymorphonuclear leukocytes. This method was applied for the analysis of samples collected from CF patients taking part to a double-blind, randomized, crossover placebo-controlled clinical trial aiming at evaluating in these patients the immunomodulatary effect of a liquid supplementation enriched in omega-3 PUFA in CF. This study has shown that omega-3 PUFA are incorporated in CF neutrophil membranes and results into a modulation of leucotrienes B production, as testified by a three fold decrease in LTB4/LTB5 ratio after omega-3 PUFA supplementation. However, no clinical improvement was observed upon omega-3 supplementation, very reproducible results observed allow to be optimistic for a future larger trial focused on clinical outcomes. In conclusion, even if the results show that omega-3 PUFA are absorbed by CF patients and that the subsequent decrease in LTB4/LTB5 ratio suggests that in such conditions, neutrophils may produce less pro-inflammatory mediators, the clinical relevance of those observations remains to be demonstrated. Future multicentric studies focusing on clinical endpoints are still warranted to determine the importance of omega-3 PUFA in CF therapeutics. RÉSUMÉ Les patients atteints de mucoviscidose (patients CF) souffrent d'infections pulmonaires récurrentes. Celles-ci provoquent un afflux permanent de neutrophiles dans le poumon, neutrophiles qui libèrent des enzymes protéolytiques et des radicaux libres responsables à long terme de la destruction du tissu pulmonaire et, finalement, de l'insuffisance respiratoire, première cause de morbidité et de mortalité chez ces patients. La réponse inflammatoire ainsi induite peut être réduite par divers traitements anti-inflammatoires, tels que corticoïdes, anti-inflammatoires non stéroïdiens ou azithromycine. L'apport oral en acides gras polyinsaturés (AGPI) oméga-3 pourrait être une autre approche thérapeutique intéressante. Ces nutriments sont décrits comme possédant des propriétés anti-inflammatoires notamment en favorisant la synthèse d'eicosanoïdes pourvus d'une activité inflammatoire moindre par rapport à ceux issus d'une autre famille d'AGPI, les oméga-6. Ce travail de thèse a pour objectif premier d'évaluer l'impact de cette approche nutritionnelle sur diverses fonctions du neutrophile chez des patients CF. Cependant un intérêt de nature prospective a également été porté à certains paramètres cliniques, afin d'évaluer la faisabilité d'une future étude axée sur des effets cliniques. Pour ce faire, une méthode de chromatographie liquide à haute performance couplée à un spectromètre de masse a été développée et validée. Cette analyse devait permettre le dosage simultané de deux eicosanoïdes, le leucotriène B4 (LTB4) issu des AGPI oméga-6 et le leucotriène B5 (LTB5) issu des AGPI oméga-3. Puis, une étude clinique, double aveugle, randomisée, croisée sans période de washout, mais contrôlée avec un placebo, a été mise au point pour évaluer l'effet immunomodulateur de ces AGPI oméga-3 donnés sous la forme d'un liquide nutritif chez des patients CF. Les résultats de cette étude ont permis de démontrer l'absorption intestinale des AGPI oméga-3 par les patients. De plus, leur administration a permis de modifier la production de teucotriène B. En effet, le ratio LTB4/LTB5 a été diminué de près de trois fois sous liquide nutritif enrichi en AGPI oméga-3. Enfin aucune différence n'a pu être notée pour les paramètres cliniques; toutefois les résultats reproductibles observés permettent d'envisager qu'une future étude multicentrique axée sur des effets cliniques est faisable. En conclusion, la modification de la composition en AGPI membranaires du neutrophile observée durant cette étude laisse penser que ces nutriments sont absorbés par les patients CF. La modulation de la production en LTBs qui en découle permet d'envisager un potentiel effet anti-inflammatoire. Toutefois, la relevance clinique de ces observations restent à être démontrée. A l'heure actuelle, une étude multicentrique, focalisée sur des paramètres cliniques, est nécessaire avant de pouvoir se prononcer sur l'utilisation des AGPI oméga-3 chez les patients CF.

Clinical and hormonal effects of the new angiotensin II receptor antagonist LRB081.

The renin-angiotensin system is a major contributor to the pathophysiology of cardiovascular diseases such as congestive heart failure and hypertension. Antagonizing angiotensin (Ang) II at the receptor site may produce fewer side effects than inhibition of the promiscuous converting enzyme. The present study was designed to assess in healthy human subjects the effect of LRB081, a new orally active AT1-receptor antagonist, on the pressor action of exogenous Ang II. At the same time, plasma hormones and drug levels were monitored. At 1-week intervals and in a double-blind randomized fashion, 8 male volunteers received three doses of LRB081 (10, 40, and 80 mg) and placebo. Blood pressure (BP) was measured at a finger by photoplethysmograph. The peak BP response to intravenous injection of a standard dose of Ang II was determined before and for < or = 24 h after administration of an oral dose of LRB081 or placebo. After drug administration, the blood BP response to Ang II was expressed in percent of the response before drug administration. At the same time, plasma renin activity (PRA), Ang II, aldosterone, catecholamine (radioassays), and drug levels (by high-performance liquid chromatography) were monitored. After LRB081 administration, a dose dependent inhibition of the BP response to Ang II was observed. Maximal inhibition of the systolic BP response was 54 +/- 3 (mean +/- SEM), 63 +/- 2, and 93 +/- 1% with 10, 40, and 80 mg LRB081, respectively. The time to peak was 3 h for 6 subjects and 4 and 6 h for 2 others. Preliminary plasma half-life (t1/2) was calculated at 2 h. With the highest dose, the inhibition remained significant for 24 h (31 +/- 5%, p < 0.05). Maximal BP-blocking effect and maximal plasma drug level coincided, suggesting that the unmetabolized LRB081 is responsible for the antagonistic effect. PRA and Ang II increased dose dependently after LRB081 intake. Aldosterone, epinephrine, and norepinephrine concentrations remained unchanged. No clinically significant adverse reaction was observed during the study. LRB081 is a well-tolerated, orally active, potent, and long-acting Ang II receptor antagonist. Unlike in the case of losartan, no active metabolite of LRB081 has been shown to be responsible for the main effects.

Plasma and cerebrospinal fluid population pharmacokinetics of temozolomide in malignant glioma patients

RESUME : Objectif: Le glioblastome multiforme (GBM) est la tumeur cérébrale maligne la plus agressive qui conduit au décès de la majorité des patients moins d'une année après le diagnostic. La plupart des agents chimiothérapeutiques actuellement disponibles ne traversent pas la barrière hémato¬encéphalique et ne peuvent par conséquent pas être utilisés pour ce type de tumeur. Le Temozolomide (TMZ) est un nouvel agent alkylant récemment développé pour le traitement des gliomes malins. A ce jour, très peu d'informations sont disponibles sur la pénétration intra-cérébrale de cet agent. Au cours d'une étude pilote de phase II menée auprès de 64 patients atteints de GBM, l'administration précoce de TMZ combinée à une radiothérapie standard (RT) afin d'intervenir au plus tôt dans l'évolution de la maladie, a permis de prolonger la survie de ces patients, résultat qui pu être confirmé par la suite lors de l'étude randomisée de phase III. L'objectif de cette étude a été de déterminer les paramètres pharmacocinétique du TMZ dans le plasma et le liquide céphalo-rachidien (LCR), d'évaluer l'influence de certains facteurs individuels (âge, sexe, surface corporelle, fonction rénale/hépatique, co-médications, RT concomitante) sur ces différents paramètres, et enfin d'explorer la relation existant entre l'exposition au TMZ et certains marqueurs cliniques d'efficacité et de toxicité. Matériel et Méthode: Les concentrations de TMZ ont été mesurées par chromatographie liquide à haute performance (HPLC) dans le plasma et le LCR de 35 patients atteints de GBM nouvellement diagnostiqués (étude pilote) ou de gliomes malins en récidive (étude récidive). L'analyse pharmacocinétique de population a été réalisée à l'aide du programme NONMEM. L'exposition systémique et cérébrale, définie par les AUC (Area Under the time-concentration Curve) dans le plasma et le LCR, a été estimée pour chaque patient et corrélée à la toxicité, la survie ainsi que la survie sans progression tumorale. Résultats: Un modèle à 1 compartiment avec une cinétique d'absorption et de transfert Kplasma -> LCR de ordre a été retenu afin de décrire le profil pharmacocinétique du TMZ. Les valeurs moyennes de population ont été de 10 L/h pour la clairance, de 30.3 L pour le volume de distribution, de 2.1 h pour la 1/2 vie d'élimination, de 5.78 hE-1 pour la constante d'absorption, de 7.2 10E4 hE-1 pour Kplasma->LCR et de 0.76 hE-1 pour KLCR plasma. La surface corporelle a montré une influence significative sur la clairance et le volume de distribution, alors que le sexe influence la clairance uniquement. L'AUC mesurée dans le LCR représente ~20% de celle du plasma et une augmentation de 15% de Kplasma->LCR a été observée lors du traitement concomitant de radiochimiothérapie. Conclusions: Cette étude est la première analyse pharmacocinétique effectuée chez l'homme permettant de quantifier la pénétration intra-cérébrale du TMZ. Le rapport AUC LCR/AUC Plasma a été de 20%. Le degré d'exposition systémique et cérébral au TMZ ne semble pas être un meilleur facteur prédictif de la survie ou de la tolérance au produit que ne l'est la dose cumulée seule. ABSTRACT Purpose: Scarce information is available on the brain penetration of temozolomide (TMZ), although this novel methylating agent is mainly used for the treatment of ma¬lignant brain tumors. The purpose was to assess TNIZ phar¬macokinetics in plasma and cerebrospinal fluid (CSF) along with its inter-individual variability, to characterize covari¬ates and to explore relationships between systemic or cere¬bral drug exposure and clinical outcomes. Experimental Design: TMZ levels were measured by high-performance liquid chromatography in plasma and CSF samples from 35 patients with newly diagnosed or recurrent malignant gliomas. The population pharmacoki¬netic analysis was performed with nonlinear mixed-effect modeling software. Drug exposure, defined by the area un¬der the concentration-time curve (AUC) in plasma and CSF, was estimated for each patient and correlated with toxicity, survival, and progression-free survival. Results: A three-compartment model with first-order absorption and transfer rates between plasma and CSF described the data appropriately. Oral clearance was 10 liter/h; volume of distribution (VD), 30.3 liters; absorption constant rate, 5.8 hE-1; elimination half-time, 2.1 h; transfer rate from plasma to CSF (Kplasma->CSF), 7.2 x 10E-4hE-1 and the backwards rate, 0.76hE-1. Body surface area signifi¬cantly influenced both clearance and VD, and clearance was sex dependent. The AU CSF corresponded to 20% of the AUCplasma. A trend toward an increased K plasma->CSF of 15% was observed in case of concomitant radiochemo-therapy. No significant correlations between AUC in plasma or CSF and toxicity, survival, or progression-free survival were apparent after deduction of dose-effect. Conclusions: This is the first human pharmacokinetic study on TMZ to quantify CSF penetration. The AUC CSF/ AUC plasma ratio was 20%. Systemic or cerebral exposures are not better predictors than the cumulative dose alone for both efficacy and safety.

HA-1 and the SMCY-derived peptide FIDSYICQV (H-Y) are immunodominant minor histocompatibility antigens after bone marrow transplantation.

BACKGROUND: Allogeneic bone marrow donors can be incompatible at different levels. Even HLA-identical pairs will be still incompatible for numerous minor histocompatibility antigens (mHag). Nevertheless, some incompatibilities are found to be associated with an increased risk of graft-versus-host disease (GVHD), which could be related to the way the immune system recognizes these antigens. METHODS: We determined the specificity of cytotoxic T-cell clones isolated during acute GVHD or during bone marrow graft rejection in patients (n=14) transplanted with marrow from donors who were histoincompatible for different minor and/or major histocompatibility antigens. RESULTS: We found a clear hierarchy among the different types of histoincompatibilities. In three combinations mismatched for a class I allele, all 27 clones isolated during GVHD were specific for the incompatible HLA molecule. In the 11 class I-identical combinations, 14 different mHags were recognized. The mHag HA-1, known to have a significant impact on the development of GVHD, was recognized in the two HA-1-incompatible combinations. In one of these combinations, which was sex mismatched, all 56 clones analyzed were directed against HA-1, demonstrating the dominance of this mHag. In the four HA-1-compatible, sex-mismatched combinations, the anti-H-Y response was directed against one immunodominant epitope rather than against multiple Y-chromosome-encoded epitopes. All male specific cytotoxic T lymphocytes (n=15) recognized the same high-performance liquid chromatography-purified peptide fraction presented by T2 cells. Moreover, all cytotoxic T lymphocytes tested (n=6) were specific for the SMCY-derived peptide FIDSYICQV, originally described as being the H-Y epitope recognized in the context of HLA-A*0201. CONCLUSIONS: Some histocompatibility antigens are recognized in an immunodominant fashion and will therefore be recognized in the majority of mismatched combinations. Only for such antigens, correlations between mismatches and the occurrence of GVHD or graft rejections will be found.

Photosensitizing effects of m-tetrahydroxyphenylchlorin on human tumor xenografts: correlation with sensitizer uptake, tumor doubling time and tumor histology.

The photodynamic effects of m-tetrahydroxyphenylchlorin (mTHPC) were assessed on human malignant mesothelioma, squamous cell carcinoma and adenocarcinoma xenografts grown in nude mice and were correlated with mTHPC uptake, histology and doubling time of the tumors. Non-thermal laser light was delivered to the tumor as surface radiation 4 days after intraperitoneal administration of 0.1 and 0.3 mg mTHPC/kg body weight, respectively. The extent of tumor necrosis was measured by histomorphometry. The mTHPC concentration in non-irradiated tumors was assessed by high-performance liquid chromatography (HPLC). The tumors were graded according to their doubling time and their vascular architecture as assessed by histology. The 0.1 mg/kg dose of mTHPC resulted in an equal uptake for all 3 tumor types but revealed a larger extent of photosensitized necrosis for adenocarcinoma, which displayed a delicate tumor stroma with numerous small capillary vessels, than for mesothelioma and squamous cell carcinoma, which were both poor in stroma and vessels. The 0.3 mg/kg dose of mTHPC resulted in a 2-fold higher tumor uptake for all 3 tumor types and in a larger extent of necrosis for mesothelioma and squamous cell carcinoma, but not for adenocarcinoma xenografts, compared with the lower drug dose. Our results demonstrate that different tumor xenografts respond differently to mTHPC-PDT for a given drug-light condition. In this setting, the photosensitizing effect was more closely related to the vascular architecture of the tumors than to the sensitizer uptake and doubling time of the different tumors

Poly-epsilon-caprolactone intravitreous devices: an in vivo study.

PURPOSE: The objective of this study was to evaluate the long-term safety and pharmacokinetic profile of a dexamethasone-loaded poly-epsilon-caprolactone (PCL) intravitreous implant. METHODS: The PCL devices were prepared by compression and were inserted into the vitreous of pigmented rabbits. At different time points, vitreous samples were retrieved, and dexamethasone concentration was analyzed by high-performance liquid chromatography. The biodegradation of the implants was evaluated by scanning electron microscopy, and the dexamethasone remaining was evaluated at the end of follow-up. Clinical and histologic examinations were performed to evaluate the implant's tolerance. RESULTS: The PCL implant allows for a controlled and prolonged delivery of dexamethasone in rabbits eyes since it released the drug within the therapeutic range for at least 55 weeks. At 55 weeks approximately 79% of the drug was still present in the implant. Biodegradation study showed that PCL implants degradation is very slow. Clinical and histologic observations showed that the devices were very well tolerated in the rabbit eye. CONCLUSIONS: This study demonstrates the feasibility and tolerance of intravitreous PCL drug delivery systems, which can offer a wide range of applications for intraocular drug delivery because of their controlled and prolonged release over months or even years.

In vivo characterisation of a novel water-soluble Cyclosporine A prodrug for the treatment of dry eye disease.

Cyclosporine A (CsA) has been demonstrated to be effective for the treatment of a variety of ophthalmological conditions, including ocular surface disorders such as the dry eye disease (DED). Since CsA is characterised by its low water solubility, the development of a topical ophthalmic formulation represents an interesting pharmaceutical question. In the present study, two different strategies to address this challenge were studied and compared: (i) a water-soluble CsA prodrug formulated within an aqueous solution and (ii) a CsA oil-in-water emulsion (Restasis, Allergan Inc., Irvine, CA). First, the prodrug formulation was shown to have an excellent ocular tolerance as well as no influence on the basal tear production; maintaining the ocular surface properties remained unchanged. Then, in order to allow in vivo investigations, a specific analytical method based on ultra high pressure liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was developed and optimised to quantify CsA in ocular tissues and fluids. The CsA ocular kinetics in lachrymal fluid for both formulations were found to be similar between 15 min and 48 h. The CsA ocular distribution study evidenced the ability of the prodrug formulation to penetrate into the eye, achieving therapeutically active CsA levels in tissues of both the anterior and posterior segments. In addition, the detailed analysis of the in vivo data using a bicompartmental model pointed out a higher bioavailability and lower elimination rate for CsA when it is generated from the prodrug than after direct application as an emulsion. The interesting in vivo properties displayed by the prodrug solution make it a safe and suitable option for the treatment of DED.

A validated assay for measuring doxorubicin in biological fluids and tissues in an isolated lung perfusion model: matrix effect and heparin interference strongly influence doxorubicin measurements.

Doxorubicin is an antineoplasic agent active against sarcoma pulmonary metastasis, but its clinical use is hampered by its myelotoxicity and its cumulative cardiotoxicity, when administered systemically. This limitation may be circumvented using the isolated lung perfusion (ILP) approach, wherein a therapeutic agent is infused locoregionally after vascular isolation of the lung. The influence of the mode of infusion (anterograde (AG): through the pulmonary artery (PA); retrograde (RG): through the pulmonary vein (PV)) on doxorubicin pharmacokinetics and lung distribution was unknown. Therefore, a simple, rapid and sensitive high-performance liquid chromatography method has been developed to quantify doxorubicin in four different biological matrices (infusion effluent, serum, tissues with low or high levels of doxorubicin). The related compound daunorubicin was used as internal standard (I.S.). Following a single-step protein precipitation of 500 microl samples with 250 microl acetone and 50 microl zinc sulfate 70% aqueous solution, the obtained supernatant was evaporated to dryness at 60 degrees C for exactly 45 min under a stream of nitrogen and the solid residue was solubilized in 200 microl of purified water. A 100 microl-volume was subjected to HPLC analysis onto a Nucleosil 100-5 microm C18 AB column equipped with a guard column (Nucleosil 100-5 microm C(6)H(5) (phenyl) end-capped) using a gradient elution of acetonitrile and 1-heptanesulfonic acid 0.2% pH 4: 15/85 at 0 min-->50/50 at 20 min-->100/0 at 22 min-->15/85 at 24 min-->15/85 at 26 min, delivered at 1 ml/min. The analytes were detected by fluorescence detection with excitation and emission wavelength set at 480 and 550 nm, respectively. The calibration curves were linear over the range of 2-1000 ng/ml for effluent and plasma matrices, and 0.1 microg/g-750 microg/g for tissues matrices. The method is precise with inter-day and intra-day relative standard deviation within 0.5 and 6.7% and accurate with inter-day and intra-day deviations between -5.4 and +7.7%. The in vitro stability in all matrices and in processed samples has been studied at -80 degrees C for 1 month, and at 4 degrees C for 48 h, respectively. During initial studies, heparin used as anticoagulant was found to profoundly influence the measurements of doxorubicin in effluents collected from animals under ILP. Moreover, the strong matrix effect observed with tissues samples indicate that it is mandatory to prepare doxorubicin calibration standard samples in biological matrices which would reflect at best the composition of samples to be analyzed. This method was successfully applied in animal studies for the analysis of effluent, serum and tissue samples collected from pigs and rats undergoing ILP.

Combining bone resorption markers and heel quantitative ultrasound to discriminate between fracture cases and control

Rapport de synthèse : L'ostéoporose est reconnue comme un problème majeur de santé publique. Comme il existe actuellement des traitements préventifs efficaces pour minimiser le risque de fracture, il est essentiel de développer des nouvelles stratégies de détection des femmes à risque de fracture. Les marqueurs spécifiques du remodelage osseux dosés dans les urines ainsi que les ultrasons quantitatifs du talon ont été étudiés comme outils cliniques pour prédire le risque fracturaire chez les femmes âgées. Il n'existe cependant que très peu de donnée sur la combinaison de ces deux outils pour améliorer la prédiction du risque de fracture. Cette étude cas-contrôle, réalisée chez 368 femmes âgées de 76 ans en moyenne d'une cohorte suisse de femmes ambulatoires, évalue la capacité discriminative entre 195 femmes avec fracture non-vertébrale à bas traumatisme et 173 femmes sans fractures - de deux marqueurs urinaires de la résorption osseuse, les pyridinolines et les deoxypyridinolines, ainsi que deux ultrasons quantitatifs du talon, le Achilles+ (GE-Lunar, Madison, USA) et le Sahara (Hologic, Waltham, USA). Les 195 patientes avec une fracture ont été choisies identiques aux 173 contrôles concernant Page, l'indice de masse corporel, le centre médical et la durée de suivi jusqu'à la fracture. Cette étude montre que les marqueurs urinaires de la résorption osseuse ont une capacité environ identique aux ultrasons quantitatifs du talon pour discriminer entre les patientes avec fracture non-vertébrale à bas traumatisme et les contrôles. La combinaison des deux tests n'est cependant pas plus performante qu'un seul test. Les résultats de cette étude peuvent aider à concevoir les futures stratégies de détection du risque fracturaire chez les femmes âgées, qui intègrent notamment des facteurs de risque cliniques, radiologiques et biochimiques. Abstract : Summary : This nested case-control analysis of a Swiss ambulatory cohort of elderly women assessed the discriminatory power of urinary markers of bone resorption and heel quantitative ultrasound for non-vertebral fractures. The tests all discriminated between cases and controls, but combining the two strategies yielded no additional relevant information. Introduction : Data are limited regarding the combination of bone resorption markers and heel quantitative bone ultrasound (QUS) in the detection of women at risk for fracture. Methods In a nested case-control analysis, we studied 368 women (mean age 76.213.2 years), 195 with low-trauma non-vertebral fractures and 173 without, matched for age, BMI, medical center, and follow-up duration, from a prospective study designed to predict fractures. Urinary total pyridinolines (PYD) and deoxypyridinolines (DPD) were measured by high performance liquid chromatography. All women underwent bone evaluations using Achilles+ and Sahara heel QUS. Results : Areas under the receiver operating-characteristic curve (AUC) for discriminative models of the fracture group, with 95% confidence intervals, were 0.62 (0.560.68) and 0.59 (0.53-0.65) for PYD and DPD, and 0.64 (0.58-0.69) and 0.65 (0.59-0.71) for Achilles+ and Sahara QUS, respectively. The combination of resorption markers and QUS added no significant discriminatory information to either measurement alone with an AUC of 0.66 (0.600.71) for Achilles+ with PYD and 0.68 (0.62-0.73) for Sahara with PYD. Conclusions : Urinary bone resorption markers and QUS are equally discriminatory between non-vertebral fracture patients and controls. However, the combination of bone resorption markers and QUS is not better than either test used alone.